Double stranded nucleic acid molecules (i.e., DNA (deoxyribonucleic acid), DNA/RNA (ribonucleic acid) and RNA/RNA) associate in a double helical configuration. This double helix structure is stabilized by hydrogen bonding between bases on opposite strands when bases are paired in a particular way (A+T/U or G+C) and hydrophobic bonding among the stacked bases. Complementary base paring (hybridization) is central to all processes involving nucleic acid.
In a basic example of hybridization, nucleic acid probes or primers are designed to bind, or “hybridize,” with a target nucleic acid, for example, DNA or RNA in a sample. One type of hybridization application, in situ hybridization (ISH), includes hybridization to a target in a specimen wherein the specimen may be in vivo, in situ, or for example, fixed or adhered to a glass slide.
The efficiency and accuracy of nucleic acid hybridization assays mostly depend on at least one of three major factors: a) denaturation (i.e., separation of, e.g., two nucleic acid strands) conditions, b) renaturation (i.e., re-annealing of, e.g., two nucleic acid strands) conditions, and c) post-hybridization washing conditions.
Traditional hybridization experiments, such as ISH assays, use a formamide-containing solution to denature doubled stranded nucleic acid. Formamide disrupts base pairing by displacing loosely and uniformly bound hydrate molecules and by causing “formamidation” of the Watson-Crick binding sites. Thus, formamide has a destabilizing effect on double stranded nucleic acids and analogs.
Once the complementary strands of nucleic acid have been separated, a “renaturation” or “reannealing” step allows the primers or probes to bind to the target nucleic acid in the sample. This step is also sometimes referred to as the “hybridization” step. The re-annealing step is by far the most time-consuming aspect of traditional hybridization applications. See FIGS. 1 and 2 (presenting examples of traditional hybridization times). In addition, the presence of formamide in a hybridization buffer can significantly prolong the renaturation time, as compared to aqueous denaturation solutions without formamide.
After the complementary strands of nucleic acid have reannealed, any unbound and mis-paired probe is removed by a series of post-hybridization washes. The specificity of the interaction between the probe and the target is largely determined by stringency of these post-hybridization washes. Duplexes containing highly complementary sequences are more resistant to high-stringency conditions than duplexes with low complementary. Thus, increased stringency conditions can be used to remove non-specific bonds between the probe and the target nucleic acids.
Four main variables are typically adjusted to influence the stringency of the post-hybridization washes:                1. Temperature (as temperature increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences).        2. Salt conditions (as salt concentration decreases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences).        3. Formamide concentration (as the amount of formamide increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences).        4. Time (as the wash time increases, non-perfect matches between the probe and the target sequence will denature, i.e., separate, before more perfectly matched sequences).        
Other factors such as pH, rate of agitation, and number of washes will also influence the stringency of the wash step. However, the use of high temperatures and/or high formamide concentrations can have significant drawbacks. For example, heat can be destructive to membranes, sample morphology, and to the nucleic acid itself. Heat can lead to complications when small volumes are used, since evaporation of aqueous buffers is difficult to control. In addition, formamide is a toxic, hazardous material, subject to strict regulations for use and waste. Furthermore, the use of a high concentration of formamide appears to cause morphological destruction of cellular, nuclear, and/or chromosomal structure.
Thus, a need exists for overcoming the drawbacks associated with the traditional post-hybridization washes of hybridization applications. By addressing this need, the present invention provides several potential advantages over prior art hybridization applications, such as increased specificity, lower background, lower wash temperatures, preservation of sample morphology, and less toxic hybridization solvents.